(a) Field of the Invention
This invention relates to insolubilized enzymes and their preparation; more especiallly the invention is concerned with a method of preparing an insolubilized, enzymatically active substance, which is applicable to a wide variety of enzymes; the invention further relates to a method of regenerating a carrier material for the enzymes, from spent, insolubilized enzyme.
(b) Description of Prior Art
Enzymes have found wide application in industry in view of their ability to initiate, promote and govern the course of a chemical or biological reaction. The enzyme can be considered as a catalyst since it does not become a part of the product formed in the reaction. On the other hand, the enzyme eventually becomes inactive or spent after taking part as a catalyst in a chemical or biological reaction.
Various techniques have been developed to produce enzymatically active substances in a physical form such that they can be used in industrial processes. The art has in general referred to such techniques as comprising the "immobilization" of the enzyme.
The objective of such immobilization may be, for example, to insolubilize the enzyme relative to a particular solvent, usually water; or to support the enzyme on an inert carrier such that a bed of enzymatically active substance is formed; or, indeed, the immobilization may be to achieve both these objectives and other objectives.
Enzymes have been immobilized on inorganic and organic carrier materials in a number of ways, for example, entrapment in polymeric gels, chemical coupling with organic or inorganic supports, microencapsulation and adsorption on various porous materials, for example glass, cellulose, activated carbon and metal oxides.
Canadian Pat. No. 688,111, Ephraim Katchalski, issued June 9, 1964, describes water-insoluble enzyme substances in which the enzyme is linked to a polymeric water insoluble carrier by a chain formed polymeric link. Canadian Pat. No. 830,477, Avraham Patchornik, issued Dec. 23, 1969, describes the bonding of an enzyme via one of its functional groups to cellulose by reaction with bromoacetyl cellulose. Canadian Pat. No. 945,921, Ralph A. Messing et al, issued Apr. 23, 1974, describes the use of a silane coupling agent to insolubilize an enzyme on an inorganic support, in which the inorganic silicon portion of the coupling agent is attached to the inorganic support and the organic portion of the coupling agent is attached to the enzyme.
The prior coupling methods in the main require the use of a coupling agent for the enzyme and carrier, which contains an appropriate functional group to react with a functional group of the enzyme which is not essential to the enzyme activity, at least in the reaction in which the enzyme is to be employed. These prior coupling methods necessitate the selection of coupling agents with particular functional groups for employment with particular enzymes.
Furthermore, in the prior immobilized enzymes, when the enzyme activity is spent and the enzyme becomes inactive, the spent enzyme and its carrier material are discarded resulting in loss of the carrier material.